Known in the art are a number of methods of immunoenzymatic analysis. Most widely employed is the method of a heterogeneous immunoenzymatic analysis (ELISA), wherein the antigen to be determined is bound to a solid phase. Such combination is effected most frequently by adsorption of an antigen from the analyzed solution directly on the surface of a polymeric material or after a preliminary coating thereof with specific antibodies. Then the adsorbed antigen is reacted with a solution of these antibodies labeled with an enxyme (conjugate) to ensure the attachment of the conjugate to the solid phase. Thereafter, the solid phase is separated from the liquid one and one of the phases is analyzed to. The concentration of the analyzed antigen can be calculated from the value of enzymatic activity (cf. U.S. Pat. No. 3,720,760). However, this method necessitates preparation of a specific conjugate for every antigen to be determined .
To ensure the versatile character of the conjugate, it is obtained with the use of anti-species antibodies. In this case, specific antibodies are added to the antigen combined with the solid phase, followed by the addition of a conjugate of an enzyme containing the anti-species antibodies which have been obtained against the species employed for the preparation of specific antibodies (British Patent No. 1,549,069).
Particular methods, wherein these general principles are employed, are distinguished by types of the enzyme employed for the preparation of a conjugate. Properties of the enzyme define to a considerable extent the possibilities of the specific ELISA method, sensitivity and costs of the analysis in the first place. Desirable properties of the enzyme are its high specific activity and stability in storage. Previously employed for these purposes were alkaline phosphate, peroxidase, .beta.-galactosidase, lysozyme, .DELTA..sub.5,3-ketosteriodisomerase, .alpha.-amylase, glucosooxidase, and some others. Most widely employed are alkaline phosphatase, peroxydase and .beta.-galactosidase.
An important characteristic of the ELISA method resides also in the type of a substrate employed for the determination of the enzymatic activity. The enzyme defines, to a considerable extent, the selection of a substrate, but the majority of enzymes allow certain variations in the substrate structure. In this case a high sensitivity of the determination of the product of substrate hydrolysis, low costs and stability thereof in storage are desirable.
The substrate for conjugates containing alkaline phosphatase is most frequently P-nitrophenylphosphate (cf. U.S. Pat. No. 3,879,262). The enzyme--alkaline phosphatase has a high specific activity, but it is insufficiently stable in storage. The product of hydrolysis of p-nitrophenylphosphate is of a light yellow colour which is substantially imperceptible by a human eye. For this reason a measuring instrument is indispensable.
Peroxidase is frequently employed for the preparation of conjugates in the ELISA method due to its low cost (cf. U.S. Pat. No. 3,791,932). However, it has a low specific activity so that the method based thereon has a low sensitivity. Furthermore, some substrates for peroxidase exhibit carcinogenic properties.
.beta.-Galactosidase has a high activity and stability in storage. p-Nitrophenyl esters are used as it's substrates, which upon hydrolysis display a light yellow colour slightly perceptible by the human eye (cf. French Patent No. 2,288,312).
Substrates or alkaline phosphates, peroxidase and .beta.-galactosidase employed in the immunoenzymatic analysis are unstable in aqueous solutions and have a high production cost, while substrates of alkaline phosphatase and .beta.-galactosidase are also unstable in a long-term storage in the dry form.
Therefore, the prior art methods for the determination of an antigen do not ensure simultaneously a high sensitivity of analysis, necessitate the use of expensive reagents for the analysis, as well as sophisticated instruments, so that they can be used only in specialized laboratories.
It is an object of the present invention to provide a method for the determination of an antigen which would have a high sensitivity, especially in a visual determination of the antigen, which would be based on the use of stable reagents and, for this reason, could be accessible to a broad range of consumers.